Device for monitoring cells

ABSTRACT

The present invention relates to methods for detection and evaluation of metabolic activity of eukaryotic and/or prokaryotic cells based upon their ability to consume dissolved oxygen. The methods utilize a luminescence detection system which makes use of the sensitivity of the luminescent emission of certain compounds to the presence of oxygen, which quenches (diminishes) the compound&#39;s luminescent emission in a concentration dependent manner. Respiring eukaryotic and/or prokaryotic cells will affect the oxygen concentration of a liquid medium in which they are immersed. Thus, this invention provides a convenient system to gather information on the presence, identification, quantification and cytotoxic activity of eukaryotic and/or prokaryotic cells by determining their effect on the oxygen concentration of the media in which they are present.

FIELD OF THE INVENTION

[0001] This application is a continuation-in-part of U.S. Ser. No.08/715,557, filed on Sep. 18, 1996, which is a continuation-in-part ofU.S. Ser. No. 08/025,899, filed on Mar. 3, 1993, which issued as U.S.Pat. No. 5,567,598 on Oct. 22, 1996, and which is continuation of U.S.Ser. No. 07/687,359, filed on Apr. 18, 1991.

BACKGROUND OF THE INVENTION DESCRIPTION OF RELATED ART

[0002] Our environment contains a multitude of microorganisms with whichwe are continuously interacting. These interactions can be beneficial,i.e., fermentations to produce wine, vinegar or antibiotics; neutral; oreven harmful, as in the case of infectious diseases. The ubiquitouspresence of these microorganisms, thus, creates a continuing need forthe detection, identification and study of the presence and metabolicactivity of such microorganisms.

[0003] While the science of microbiology has changed significantly inthe last 25 years, many procedures for the detection, identification andanalysis of the behavior of microorganisms are still time consuming. Forexample, in the area of antimicrobic susceptibility testing nearly halfof all testing in hospitals in the United States still use theBauer-Kirby Disc Method. This method uses the presence or absence ofvisible growth of the microorganisms to indicate the efficacy of anantimicrobic compound, and generally requires an 18 to 24 hourincubation period to allow for microorganism growth before a result canbe obtained. A decrease in the time required to obtain such antimicrobicsusceptibility information is needed.

[0004] Another popular method for antimicrobic susceptibility testing isthe broth micro-dilution method, such as the Sceptor® System foridentification and antimicrobic susceptibility testing or organisms(Becton Dickinson Diagnostic Instrumentation Systems, Sparks, Md.). Thesystem uses a disposable plastic panel having a plurality of low volumecupulas (ca. 0.4 ml per cupula), each containing a different testcompound or a different concentration of a test compound dried on thecupula surface. The organism to be tested is suspended in the desiredtesting medium, and aliquots are delivered to the individual cupulas ofthe test panel. The reagent dried on the panel dissolves in the sample,and the system is then incubated overnight (18 to 24 hrs.) to allowsufficient time for the organisms to interact with the reagent and forvisible growth to appear. The panel is subsequently examined visuallyfor the presence or absence of growth, thereby obtaining information onthe susceptibility of the organism undergoing testing. Additional wellsaid in identifying the organism. However, this test method suffers fromthe drawback of also requiring a long incubation period.

[0005] One approach to the reduction of the incubation time is tomonitor metabolic activity of the microorganisms, rather than growth ofcolonies. Many approaches have been reported in the attempt to rapidlyand accurately monitor such metabolic activity.

[0006] For example, apparatus utilizing light scattering optical meanshave been used to determine susceptibility by probing the change in sizeor number of microorganisms in the presence of various antimicrobiccompounds. Commercial instruments utilizing these principles areexemplified by the Vitec System (BioMerieux Corp.). This system claimsto yield information on antimicrobic susceptibility of microorganismswithin 6 hours for many organism and drug combinations. Othercombinations can require as long as 18 hours before the antimicrobicsusceptibility of the organism can be determined by this machine.

[0007] Additionally, modifications of the Bauer-Kirby procedure havebeen developed which allow certain samples to be read in four to sixhours. However, such a system is “destructive” in nature, requiring thespraying of a developing solution of a color forming dye onto the testplate. Re-incubation and reading at a later time is, thus, not possibleand if the rapid technique fails, the experiment cannot be continued fora standard evaluation at a later time.

[0008] A bioluminescent method based on the quantity of ATP present inmultiplying organisms has been described as yielding results ofantimicrobic susceptibility testing in four and half hours for certaincompositions (Wheat et al.). However, the procedure tends to becumbersome and broad applicability has not been shown.

[0009] Other approaches have involved monitoring of microbial oxygenconsumption by the measurement of pH and/or hemoglobin color change, orby the use of dyes such as triphenyltetrazolium chloride and resazurin,that change color in response to the total redox potential of the liquidtest medium.

[0010] The monitoring of the consumption of dissolved oxygen bymicroorganisms, as a marker of their metabolism, has been studied formany years. For example, C. E. Clifton monitored the oxygen consumptionof microorganisms over a period of several days using a Warburg flask in1937. This method measured the change in oxygen concentration in a slowand cumbersome manner.

[0011] The “Clark” electrode, a newer electrochemical device, is alsocommonly used to measure dissolved oxygen. Unfortunately, the Clarkelectrode consumes oxygen during use (thereby reducing the oxygenavailable to the microorganisms) and the “standard” size electrode istypically used only to measure volumes of 100 mls or greater to preventthe electrode from interfering with the measurements.

[0012] A “miniature” Clark electrode has been described, but thiselectrode is a complicated multi-component part which must, also, be incontact with the solution to be measured. While an oxygen permeablemembrane can be used to prevent the electrode components of the devicefrom interacting with the constituents of the test solution, the oxygenmust still equilibrate between the test solution and the measurementsystem and is consumed once it passes the membrane.

[0013] Optical systems which can yield oxygen concentration data, havebeen developed to overcome the shortcomings of the Clark electrodesystems. The main advantage of such optical methods is that theinstrumentation required to determine quantitative value does not itselfmake physical contact with the test solution. Optical techniquesallowing both colorimetric and fluorometric analyses for oxygen to becarried out rapidly and reproducibly are known, and costs for suchanalyses are often quite low. For example, several luminescenttechniques for the determination of oxygen have been described which arebased on the ability of oxygen to quench the fluorescence orphosphorescence emissions of a variety of compounds. However, suchmethods have not been adapted to microbial monitoring or prokaryotic oreukaryotic cell monitoring.

[0014] Other systems have been described that provide information on thepresence, identity and antimicrobic susceptibility of microorganisms ina period of eight hours or less. Wilkins and Stones in U.S. Pat. No.4,200,493 disclose a system that uses electrodes and a high impedancepotentiometer to determine the presence of microorganisms. In U.S. Pat.No. 3,907,646 Wilkins et al. disclose an analytical method whichutilizes the pressure changes in the headspace over a flask associatedwith microbial growth for the detection and surveillance of theorganisms. U.S. Pat. No. 4,220,715 to Ahnell, discloses a system whereinthe head space gas above a test sample is passed through an externaloxygen detector for determination of the presence of microorganisms.Ahnell, in U.S. Pat. No. 4,152,213, discloses a system for analysis bymonitoring the vacuum produced by growing organisms in a closed headspace above a test sample. U.S. Pat. No. 4,116,775 to Charles et al. isan example of the use of optical means based on the increase inturbidity or optical density of a growing microbial culture for thedetection and monitoring of bacterial growth. A combined electro-opticalmeasurement of birefringence of a test solution containingmicroorganisms is described in EPO 0092958 (Lowe and Meltzer).

[0015] The increased incidence of tuberculosis and the recent emergenceof Multiple Drug Resistant (MDR) strains threatens the ability tocontrol this disease. Therefore, when a strain is resistant to two ormore drugs, such as rifampin and isoniazid, the course of treatmentincreases from 6 months to 24 months, and the cure rate decreases fromalmost 100% to less than 60%.

[0016]Mycobactetium tuberculosis (TB) is a slow growing species.Generally, at least three to five weeks of growth on solid or liquidmedia are required to produce enough cell mass for identification andsusceptibility testing. The most commonly used susceptibility method forTB is the Modified Proportion Method (NCCLS M24-T). This method requiresan additional three to four weeks of growth before the results areavailable. The total elapsed time for a find report is typically twomonths and may be as much as three months.

[0017] The BACTEC 460 instrument (Becton, Dickinson and Company,Franklin Lakes, N.J.) can reduce these times considerably. The BACTECmethod detects the presence of mycobacteria by their production ofradioactive CO₂. The BACTEC system can also detect resistant organismsby their continuing production of radioactive CO₂ in the presence ofantimycobacterial drugs.

[0018] It becomes apparent that a wide variety of methods have beenapplied to the detection and the antibiotic susceptibility testing ofmicroorganisms. Many of these methods can only yield useful data whenmonitored by instruments dedicated to this task. Thus there exists aneed for a system which can allow determinations of the presence andbehavior of microorganisms without the requirement of dedicatedinstrumentation. Further there exists a need for a system that willallow the determination of the effect of a compound such as anantibiotic on a sample of microorganisms in a short time that does notsignificantly alter the behavior of the microorganisms.

[0019] There also currently exists a need for improved methods ofmeasuring eukaryotic and/or prokaryotic cell growth and viability, suchas, for example, in the areas of drug discovery and development. Animportant application for these methods is in testing and quantifyingthe effects of therapeutic drugs, drug candidates, toxins and chemicalson the growth of cell lines (i.e, cytotoxicity assays). As an example,potential chemotherapeutic drug candidates are frequently tested at anumber of concentrations to determine their potency for inhibiting thegrowth of selected mammalian tumor cell lines.

[0020] The most commonly used reagent for eukaryotic (i.e., mammalian)cell cytotoxicity assays is MTT(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) [“RapidColorimetric Assay for Cellular Growth and Survival: Application toProliferation and Cytotoxicity Assays”, T. Mossmann, J. Immunol. Methods(1983), vol. 65, 55-63]. This tetrazolium salt is reduced within themitochondria of metabolically active cells to form a colored precipitate(formazan dye). For cytotoxicty measurements, the cells are typicallygrown in a microwell trays containing various concentrations of drug.MTT is added and incubated with cells for 1-4 hours, the cells arelysed, the formazan dye is resolubilized by thorough mixing and adose-response curve is obtained from endpoint absorbance measurements.Among disadvantages of this method are the multiple reagent additionswhich are required. MTT is also susceptible to interferences from somedrugs with reducing groups and from precipitation of some drugs,especially those adsorbing light in the visible region. The test itselfis non-reversible and further time point readings of the same cellcultures cannot be performed without setting up a separate assay to beused for each time point.

[0021] Another redox indicator suggested for cytotoxicity assays isresazurin which is reduced to resorufin in the presence of growingcells. Resazurin is subject to autoreduction in some media which cancause false positive signals. An improved formulation of resazurin witha redox stabilizing buffer known as “Alamar Blue” has been introduced tosolve this autoreduction problem in U.S. Pat. No. 5,501,959. Thisformulation, however, still requires the addition of dye and buffer tothe cells and is essentially a non-reversible reduction.

[0022] Another method for determining cell viability is to measureuptake of radiolabeled nucleotides such as tritiated thymidine. Thistest is very sensitive but it is relatively expensive, time-consuming,and requires multiple steps. It also requires the handling and disposalof radioisotopic waste. This type of assay cannot readily be automatedor adapted to formats for rapid drug screening purposes.

SUMMARY OF THE INVENTION

[0023] It is therefore an object of this invention to provide animproved means to detect the presence of, and to evaluate the metabolicactivity of, eukaryotic cells present in a liquid or semi-solid media.It is further an object of this invention to provide a microbialmonitoring device or system which can be simply read and visuallyinterpreted, and which permits results to be obtained in a shorter timeperiod than previously attainable, nominally 6 hours or less.Additionally, it is an object of the invention to provide a means fordetection and/or monitoring the activity of eukaryotic cells without theuse of dedicated instrumentation.

[0024] The above and related objects are realized by the processes ofthe instant invention. These processes utilize a luminescence detectionsystem, and more particularly, a fluorescence detection system whereinthe fluorescing sensor compound is one which exhibits a quantifiabledegree of quenching when exposed to oxygen. In one embodiment, thesensor compound may be brought into contact with the test sample (eitherdirectly or separated by an oxygen permeable membrane) and thefluorescence is measured or observed visually with appropriate aids. Inanother embodiment, an increase in fluorescence is indicative ofrespiring aerobic microorganisms, which utilize (and thereby reduce) theoxygen in the sample.

[0025] The sensor need not be in direct contact with the test sample.The only requirement is that the test sample and sensor are in acontainer substantially isolated from atmospheric oxygen so that thesensor can react to the presence/absence of oxygen in the container.

[0026] The system can, thus, be used to detect a variety of respiringeukaryotic and/or prokaryotic cells and can be used in cytoxicity assaysfor the effects of drugs, toxins, or chemicals on eukaryotic and/orprokaryotic cells.

BRIEF DESCRIPTION OF THE FIGURES

[0027]FIG. 1 graphically depicts intensity of fluorescence as a functionof time for indicators in contact with broth containing organisms andbroth containing no organisms.

[0028]FIG. 2 graphically depicts the intensity of fluorescence as afunction of time for indicators in contact with broth inoculated withdifferent concentrations of microorganisms.

[0029]FIG. 3 graphically depicts the intensity of fluorescence as afunction of time for indicators in contact with broth inoculated withthe same number of organisms but containing different concentrations ofphenol.

[0030]FIG. 4 graphically depicts the intensity of fluorescence as afunction of time for indicators in contact with broth inoculated withthe same number of organisms but containing different amounts of coppersulfate.

[0031]FIG. 5A graphically depicts the fluorescence, as a function ofindicators in contact with broth inoculated with the same concentrationof microorganisms but different concentrations of cefuroxime. Some wellswere covered with mineral oil to prevent oxygen from diffusing into thewells. The fluorescence is given as a percent of growth control.

[0032]FIG. 5B graphically depicts the fluorescence as a percent of thegrowth control in wells that are overlaid with oil or left open andmeasured at several different times.

[0033]FIG. 6 graphically depicts the intensity of fluorescence ofindicators in blood culture bottles when measured continuously over 16hours. The arrows indicate the times when samples were removed in orderto quantify the concentration of organisms present.

[0034]FIG. 7 depicts the data collected in the BACTEC® instrumentindicating the change in fluorescence intensity indicative of the growthof P. aeruginosa.

[0035]FIG. 8 depicts the data collected in the BACTEC® instrumentindicating the change in fluorescence intensity indicative of the growthof M. fortuitum.

[0036]FIG. 9 depicts the data collected in the BACTEC® instrumentindicating the change in fluorescence intensity indicative of the growthof E. coli.

[0037]FIG. 10 graphically depicts intensity of fluorescence by plottingfluorescence signal vs. cell number for HL60 cells growing in oxygensensor plates, wherein the cell number was determined by averagedhemacytometer readings.

[0038]FIG. 11 graphically depicts intensity of fluorescence by plottingfluorescence signal vs. the initial cell number for U937 cells grown inoxygen sensor plates.

[0039]FIG. 12 graphically depicts intensity of fluorescence by plottingfluorescence vs. concentration of vinblastine in a cytotoxicity assaywith HL60 cells at selected time points in an oxygen sensor plate.

[0040]FIG. 13 graphically depicts absorbance, by plotting absorbance vs.concentration of vinblastine in a cytotoxicity assay using MTT with HL60cells.

[0041]FIG. 14 graphically depicts intensity of fluorescence by plottingfluorescence vs. concentration of methotrexate in a cytotoxicity assaywith HL60 cells at selected time points in an oxygen sensor plate.

[0042]FIG. 15 graphically depicts intensity of fluorescence by plottingfluorescence vs. concentration of sodium azide in a cytotoxicity assaywith HL60 cells at selected time points in an oxygen sensor plate.

[0043]FIG. 16 graphically depicts intensity of fluorescence by plottingfluorescence vs. concentration of SDS in a cytotoxicity assay with HL60cells at selected time points in an oxygen sensor plate.

[0044]FIG. 17 graphically depicts intensity of fluorescence by plottingfluorescence vs. time for oxygen sensor plates in which MCD-1 cells weregrown on the indicated amounts of MATRIGEL®.

[0045]FIGS. 18A, 18B and 18C graphically depict intensity offluorescence by plotting fluorescence vs. time for oxygen sensor platesin which MCD-1, SK-N-SH, and NIH3T3 cell lines were grown on MATRIGEL®.

[0046]FIG. 19A graphically depicts relative changes in the intensity offluorescence over time for HL60 cells grown in a 24-well plate (i) withthe sensor on the bottom of the well (unmodified RuSi); (ii) with thesensor on the bottom of an insert. The third data set is for a controlof media only with no cells. FIG. 19B shows the same experiments as inFIG. 19A, but using adherent cell line MDCK instead of HL60 cells.

[0047]FIG. 20 graphically depicts the normalized fluorescence intensityvs. the number of HL60 cells, grown in 96 well oxygen sensor plates andin 384 well sensor plates.

[0048]FIG. 21 graphically depicts relative changes in the intensity offluorescence by plotting fluorescence vs. time for SF-9 insect cellsgrown at the indicated concentrations in a 96 well oxygen sensor plate.

[0049]FIG. 22 graphically depicts relative changes in the intensity offluorescence by plotting fluorescence vs. time for yeast cells grown atthe indicated concentrations in a 96 well oxygen sensor plate.

[0050]FIG. 23 graphically depicts the relationship between the initialconcentration of yeast cells and the time required to reach 120% of theinitial fluorescence signal for yeast growing in media with the fourindicated glucoase concentrations.

DETAILED DESCRIPTION OF THE INVENTION

[0051] The process of this invention presents a quick, easy, andunambiguous method for the measurement and/or detection of respiringaerobic microorganisms and more generally, eukaryotic and/or prokaryoticcells, by measurement or visual observation of luminescence. The termluminescence is intended to include fluorescence and phosphorescence, aswell as time-resolved fluorescence and fluorescence lifetime. In apreferred embodiment the luminescent sensor compound can be afluorescent sensor compound. In the process of the present invention,this compound is irradiated with light containing wavelengths whichcause it to fluoresce, and the fluorescence is measured by any standardmeans, or evaluated visually.

[0052] The fluorescent compound must be one which exhibits a largequenching upon exposure to oxygen at concentration ordinarily found inthe test liquids (generally 0.4%). While virtually any such compound canbe used, preferred fluorescent compounds of this invention aretris-2,2′-bipyridyl ruthenium (II) salts, especially the chloridehexahydrate salt (Ru(BiPy)₃Cl₂), tris-4,7-diphenyl-1,10 -phenanthrolineruthenium (II) salts, especially the chloride (salt Ru(DPP)₃Cl₂), and9,10-diphenyl anthracene (DPA).

[0053] The fluorescent compound must be placed in chemical communicationwith the oxygen of the test sample to exhibit the quenching. This can beachieved by placing the compound directly in contact with the sample.However, in a preferred embodiment the compound and sample are separatedfrom each other by the interposition of a membrane embedding materialpermeable to oxygen, and relatively impermeable to the other samplecomponents, between them, thereby preventing the interaction of thesample and the compound. Neither the fluorescent compound nor themembrane in which the fluorescent compound is embedded need be in directcontact with the test sample, broth, or fluid (the compound and samplemust be substantially isolated from atmospheric oxygen, therebypreventing any false reading due to the presence of atmospheric oxygen),but still permitting reaction of the compound to the presence or absenceof oxygen as a result of respiration of microorganisms.

[0054] The system can be allowed to interact unobserved for apredetermined amount of time after which the presence or absence offluorescence is observed and compared to appropriate control samples,yielding results that are often obtained with a single such observation.A particular benefit of this system, is that the measurement offluorescence is non-destructive and if after a period of time (e.g. 4hours) the results are non-conclusive, the system can be re-incubatedand read again at a later time. Further, while it is anticipated thatthe results will be compared with reagent controls, such is by no meansnecessary, and it is postulated that, by appropriate choice offluorescent compounds, a skilled technician or technologist would becapable of independently determining whether the results indicate thepresence of microbial activity.

[0055] The detection of fluorescent intensity can be performed by anymeans ordinarily used for such measurements, e.g. a fluorometer.Alternatively, the fluorescent intensity can be observed visually and,optionally, compared with a reagent control (e.g. a system containing nolive organisms or a system with no added test chemicals). Thus, themethods can be utilized to both provide a quantitative measurement ofrelative activity, using a fluorometer, or a more qualitative estimateof such activity, by visual inspection.

[0056] In a preferred embodiment of this invention, the fluorescentcompound is chosen such that it will exhibit little or no fluorescencein the presence of oxygen. This obviates the need for a control, as theperson performing the test would interpret any appreciable fluorescence(i.e. beyond that of any nominal background fluorescence) as indicativeof the presence of microbial activity. Such results can be obtained by afluorometer or other measurement means, or preferably, visualinspection, and provide a quick, qualitative estimate of such activity.Preferred fluorescent compounds for this embodiment include Ru(BiPy)₃Cl₂and Ru(DPP)₃Cl₂.

[0057] It has also been found that for systems where the compound orcompound embedded membrane is in contact with the fluid, test sample, orbroth, while the test can be run in systems isolated from atmosphericoxygen, accurate results can also be obtained when the system is leftexposed to atmospheric oxygen. In fact, this is desirable when theorganisms are to be incubated for periods of time exceeding 2 hours, asthey would otherwise tend to consume all the dissolved oxygen in thesystem and subsequently generate a false reading. Thus, the system ofthis invention is quite versatile, and can be used in a wide array ofconditions.

[0058] A further benefit of the instant invention is that a unitizedapparatus can be constructed. Briefly, the apparatus comprises a samplecontaining reservoir, or more commonly a plurality of identicalreservoirs adapted to contain a test sample and other such liquid andsoluble components (e.g. nutrients, etc.) as may be required by theparticular application. The reservoirs also provide a luminescentindicator element which monitors the oxygen levels of the solution. Theindicator element of this invention uses a luminescent compound known toshow a large quenching of its luminescent emission when exposed tooxygen.

[0059] In a preferred embodiment of this invention, the luminescentcompound can be mixed and distributed throughout a plastic or rubberphase that is permeable to oxygen gas but relatively impermeable towater and non-gaseous solutes. Silicone rubber is a particularly usefulmaterial for this application. When a test solution containing, forexample, microorganisms, is placed in such a sample reservoir, themetabolic activity of the organisms causes a reduction in the level ofdissolved oxygen in the sample, and the sample will yield a higherluminescent signal upon excitation. Sample liquids not containingmicroorganisms will not show a decrease in their oxygen levels and willonly show low levels of luminescent due to high oxygen quenching ofluminescence.

[0060] Alternatively, the oxygen sensitive fluorophore or luminescentcompound can be in a microencapsulated form or in the form of granulesof an oxygen permeable material. It is also anticipated that thefluorophore or luminescent compound can be contained within a separatelymanufactured component such as a bead, disc, or prongs, which can beseparately introduced into the test solution. The use of prongs isparticularly advantageous as such prongs can be attached to a lid orother device to permit easy manipulation. In a preferred embodiment, aplurality of prongs can be attached to a single membrane, or other coverand thereby be maintained in an appropriate orientation such that theycan simultaneously be placed into the reservoirs of a base containing aplurality of sample reservoirs. By choice of appropriate materials, theprongs can be made impermeable to the indicator molecules and tomicroorganisms in the sample, but permeable to oxygen.

[0061] The fluorophore or luminescent compound can also be in a liquidphase separated from the solution being analyzed by a membrane that isimpermeable to the indicator molecules and to microorganisms in thesample but which is permeable to oxygen. Additionally, less-sensitivesensors can be fabricated by using less O₂ permeable polymers or byusing compounds with shorter excited-state lifetimes.

[0062] It is also considered that the luminescent sensor compound, whichis an oxygen sensor, can be a phosphorescent compound such as platinum(II) and palladium (II) octaethyl porphyrin complexes immobilized inPMMA (polymethyl methacrylate); CAB (cellulose acetate brityrate);platinum (II) and palladium (II) octaethyl porphyrin ketone complexesimmobilized in PVC (polyvinylchloride) and polystyrene.

[0063] Further, the methods of this invention can be used to test thesusceptibility of a microorganism or eukaryotic and/or prokaryotic cellsto a compound, such as an antibiotic, which is capable of severelyinhibiting the growth and/or the metabolic activity of organisms. Theincrease in luminescent signal normally caused by the organism will besuppressed in the presence of such compounds. The behavior of theluminescent signal from a reservoir will demonstrate the ability of thetest component to negatively effect the normal oxygen consumption of theorganism added to the reservoir.

[0064] In addition, any of the embodiments discussed above may beutilized so that the sensor, luminescent compound, or the membrane inwhich it is embedded need not be in direct contact with the test sample,fluid, or broth in which the microorganisms or eukaryotic and/orprokaryotic cells may be present. In such case, the sensor, compound ormembrane in which it is embedded need only be in the same container withthe test sample, fluid or broth and that they be substantially isolatedfrom atmospheric oxygen to function as an indicator of the presence orabsence of respiring microorganisms, or eukaryotic and/or prokaryoticcells.

[0065] It is also apparent that an assay method which is reversible,non-destructive to cells, requires no reagent additions, and poses noadditional disposal requirements would be advantageous for cytotoxicdrug screening, cellular quantitation, and viability testing.

[0066] The present invention describes a method for analyzing andquantifying eukaryotic and/or prokaryotic cells, and, in a preferredembodiment, mammalian cells, based on their consumption of oxygen.Examples of prokaryotes include bacteria and cyanobacteria. This groupincludes common bacteria such as Escherichia coli, widely used ingenetic engineering, and both pathogenic and non-pathogenic organismssuch as Mycobacteria, Staphylococcus, and Salmonella. Examples ofeukayotes include protists, fungi, plants and animals. This wouldinclude predominantly unicellular organisms, such as yeast and fungi,and multicellular organisms such as insects, reptiles, birds, andmammals. In a preferred embodiment, this includes cells from rodents andfrom humans and the cell lines derived from them. These examples are notintended to restrict in any way the types of cells that can be analyzedby the present invention.

[0067] Optical sensors for determining oxygen concentration based onoxygen's quenching of luminescence have been previously described[“Determination of oxygen concentrations by luminescence quenching of apolymer-immobilized transition-metal complex”, Bacon, J. R.; Demas, J.N., Anal. Chem. (1987), 59(23), 2780-5]. These consist of a luminescentdye which is sensitive to oxygen quenching immobilized in anoxygen-permeable membrane. When placed in contact with a liquid mediasuch sensors can respond to changes in oxygen content in the media dueto cellular respiration. Consumption of oxygen by living cells in asolution (or fluid or liquid media) decreases the concentration ofoxygen within a sensor in contact with the solution. The dye'sluminescence increases as the cells consume available oxygen. Suchsensors have not previously been described for determining the growth orviability of eukaryotic and/or prokaryotic cells.

[0068] Furthermore, the sensor need not be in direct contact with thesolution in order to analyze and quantify eukaryotic and/or prokaryoticcells. The only requirement is that the solution and sensor are in acontained area substantially isolated from atmospheric oxygen so thatthe sensor can react to the presence or absence of oxygen in thecontained area.

[0069] The present invention describes the use of such oxygen sensors ina microwell tray format for quantitation of eukaryotic and/orprokaryotic cell cultures, and preferably, mammalian cell cultures, andfor cytotoxicity assays. The microwell format enables reading withroutine luminescence plate readers. This format offers ease of use in anon-destructive assay in which no additional reagents are required. Thisfeature allows cells to be repeatedly monitored since no dyes orindicators are added to or released into the cellular media. The cellsgrown and monitored in the wells may thus be removed and used foradditional assays or subculturing if desired. Because this method isreadily adapted to microwell tray formats such as 96 well and 384 wellplates, the method is especially useful for high throughput screening ofdrugs, toxins and other chemicals to determine their cytotoxic activity.

[0070] Examples of drugs and toxins which can be utilized in the processof the present invention gallium nitrate, procarbazine, fludarabine,vinblastine, streptozotocin, pentostatin, mitoxantrone, hydroxyurea,piperazinedione, MGBG, 5-azacytidine, bisantrene, cytarabin, colchicine,cladribin, amsacrine, 6-thioguanine, aclarubicin, cisplatin,5-fluorourocil, bleomycin, mitomycin C, actinomycin D, methotrexate,mechlorethamine, melphalan, docetaxel, epirubicin, etoposide,vincristin, doxorubicin, teniposide, trimetrexate, topotecan, CPT 11,paclitaxel, gemcitabin, thymidine, acivicin, spirogermanium,cyclocytidine, zinostatin, flavone acctate, diglycoaldehyde,deazauridine, anguidine, PALA, aphidicolin, L-alanosine, maytansine,DQ-1, camptothecin, cremophor EL, homoharringtonine, sodium azide, DQ-2,and HgCl₂, but this is not intended to be limited to such drugs andtoxins and can include any drug or toxin which can be utilized in thepresent invention.

[0071] Examples of chemicals, including components, compounds, aminoacids, vitamins, salts, proteins and others, which can be utilized inthe process of the present invention include magnesium chloride,glucose, D-galactose, L-valine, glutamine, phenylalanine, arginine,cystine, glutamine, histidine, isoleucine, leucine, lysine, methionine,threonine, tryptophan, tyrosine, valine, biotin, choline, folate,nicotinamide, pantothenate, pyridoxal, thiamine, riboflavin, sodiumchloride, potassium chloride, NaH₂PO₄, NaHCO₃, calcium chloride,insulin, transferrin, and specific growth factors such as recombinanthuman epidermal growth factor, hydrocortisone, fibroblast growth factor,vascular endothelial growth factor, ascorbic acid (vitamin C),insulin-like growth factor and heparin, but this is not intended to belimited to such chemicals and can include any chemical which can beutilized in the present invention.

[0072] In a preferred embodiment, the oxygen sensor plates were preparedusing 96 well microtiter plates following general methods describedherein. These plates used the fluorescent dye1,7-diphenyl-1,10-phenanthroline ruthenium (II) chloride adsorbed tosilica gel and embedded in a silicone matrix.

[0073] The sensor plates may be used to quantify, for example, thenumber of viable eukaryotic cells in media using a standard microplatefluorimeter. Results are shown in FIG. 10 for a preferred embodiment forquantifying mammalian cells, for absolute number of cells during agrowth assay vs. the normalized fluorescent signal (the relative signalfor each well at a given time point divided by the initial fluorescenceof the well is referred to as “normalized fluorescence”). The absolutecell number was determined by hemacytometer at the time of each reading.An alternative quantitation method is shown in FIG. 11 where the initialcell number (i.e. “seeded cell number”) may be inferred from theincrease in normalized fluorescence over time.

[0074]FIGS. 12 and 14-16 demonstrate selected cytotoxicity assays withthe oxygen sensor plates in which an equivalent number of cells in mediawere distributed in each well. Serial dilutions of selected drugs andtoxins were prepared and added to the wells. Incubation of the platesfor the indicated periods of time produced dose-response curves fromwhich IC₅₀ results can be obtained. To demonstrate functionalequivalence to existing cytotxicity assays, parallel experiments wereperformed using MTT to obtain dose-reponse curves for the same set ofdrugs. An example, the MTT dose-response curve of vinblastine with HL60cells, is shown in FIG. 13. It is important to note that the MTT data inFIG. 13 required a separate plate for each of the timepoint curves. Theanalogous oxygen sensor assay in FIG. 12 demonstrates repeated readingsof the same sensor plate to obtain an optimal dose-response curve.

[0075] Another feature of these sensor plates is that they may becombined with additional biomaterials such as extracellular matrices.Assays using the matrix MATRIGEL® with various cell lines are shown inFIGS. 17-18. These suggest the oxygen sensor plates may be combined withextracellular matrices to gain information on both oxygen consumptionand cellular migration through the extracellular matrix.

[0076] A number of cell lines have been tested in the oxygen sensorplates under various conditions as indicated in Table 11 demonstratingthe broad applicability of this method to mammalian cell monitoring.

[0077] Although the described plates use the luminescent dye4,7-diphenyl-1,10 -phenanthroline ruthenium (II) chloride, otherluminescent dyes which exhibit significant oxygen quenching such astris-2,2′-bipyridyl ruthenium chloride may also be used. A wide varietyof silicone rubber polymers and other oxygen permeable polymers may beused to construct the sensors. Sensors may be constructed from sol-gelfilms [“Tailoring of Sol-Gel Films for Optical Sensing of Oxygen in Gasand Aqueous Phase”, C. McDonaugh, B. D. MacCraith, and A. K. McElvoy,Anal. Chem. (1998), vol. 70, 45-50]. Alternatively, useful oxygensensors can be constructed with the dye covalently immobilized tomaterials such as controlled pore glass [“Oxygen Sensing in Porous Glasswith Covalently Bound Luminescent Ru(II) Complexes”, M. P. Xavier, etal, Anal. Chem. (1998), vol. 70, 5184-5189]. Other formats may includeadding the sensor to unmodified plates in the form of beads or prongs[see U.S. Pat. No. 5,567,598].

EXAMPLES

[0078] The following examples illustrate certain preferred embodimentsof the instant invention but are not intended to be illustrative of allembodiments.

EXAMPLE 1 Preparation of an O₂-Sensitive Indicator Microtitration Tray

[0079] The fluorescent compound tris 4,7-diphenyl-1,10-phenanthrolineruthenium (II) chloride (Ru(DPP)₃Cl₂) was synthesized using theprocedure of Wafts and Crosby (J.Am.Chem. Soc. 93, 3184(1971)). A totalof 3.6 mg of the compound was dissolved in 2.0 ml dimethyl sulfoxide(D-5879, Sigma Chemical, St. Louis, Mo.) and the resultant solution wasthen added slowly, with stirring, to 1300 ml silicone rubber formingsolution (Water Based Emulsion #3-5024, Dow Corning, Midland, Mich.). A35 microliter aliquot of the mixture was subsequently dispensed intoeach well of a 96 well, flat bottom, white microtiter tray(#011-010-7901, Dynatech, Chantilly, Va.), and the system wassubsequently cured overnight in a low humidity (less than 25% RH), 60°C. incubator. After curing, the trays were washed by either soaking orby filling and emptying each well several times with each of thefollowing reagents; a) absolute ethanol, b)0.1 M phosphate buffer pH7.2, c) hot distilled water (about 45° C.) and d) ambient temperaturedistilled water.

[0080] Subsequently, 150 microliters of a Broth A, consisting of 35%Mueller Hinton II (BBL #124322, BD Microbiology Systems, CockeysvilleMd.), 15% Brucella (BDL #11088), and 50% distilled water, was dispensedinto each well of the tray, and the tray was then placed in a glove boxcontaining the desired concentration of oxygen, mixed with nitrogen toobtain a total pressure of 1 atm. The tray was kept in the glove box forat least 24 hours, after which it was covered with an adhesive backedmylar sheet and removed.

[0081] The fluorescent emissions of the fluorescent compound in thebottom of each well of the tray were then measured using a Perkin-ElmerLS-5B equipped with a microtiter reader attachment at the followinginstrument settings: 485nm excitation wavelength, 550 nm cut-on filterin the emission window, 10 nm excitation slit, and a 5 nm emission slit.The results are presented in Table 1. TABLE 1 Fluorescence of TrayEquilibrated with Various Oxygen Gas Levels % Oxygen in Mixture TrayAverage Reading (balance Nitrogen) 1 803 0.00 2 759 0.28 3 738 0.53 4524 2.45 5 484 3.40 6 445 5.35 7 208 20.90

[0082] As shown, it can be observed that indicators in wellsequilibrated in atmospheric air (Tray 7) displayed a much lowerfluorescent signal than wells equilibrated with gas mixtures containinglower concentrations of oxygen (Trays 1-6). This indicates that thefluorescent emission of the fluorescent indicator compound embedded inthe silicone rubber is related to oxygen concentration and that thesystem can be easily equilibrated with changing oxygen levels. Thesystem allowed 96 sample wells (containing 0.1-0.3 ml sample) to becontained in a single unit that is easily manipulated.

EXAMPLE 2 Use of Indicator System to Measure Relative O₂ ConcentrationProduced by a Reducing Agent

[0083] The O₂ concentration in wells of an Indicator Microtiter trayproduced as in Example 1 was varied by the addition of a strong reducingagent, sodium sulfite (which reduces O₂ content). A 150 microliteraliquot of the reducing agent (at concentrations ranging from 0 to 1083parts per million (ppm) sulfite ion in water) was pipetted into wells ofthe tray. Each well was allowed to react for 30 minutes, open to theatmosphere, and the fluorescence of the indicators measured in aFluoroskan II Fluorometer (Flow Laboratories, McLean, Va.), having anexcitation bandpass filter at a wavelength of 460 nm and an emissioncut-on filter at 570 nm. The results are presented in Table 2. TABLE 2Effect of Sodium Sulfite on Fluorescence ppm sulfite ion FluorescenceIntensity* 0 3090 65 3513 163 3545 325 4033 542 11571 1083 11863

[0084] As shown, the wells containing the highest concentrations ofreducing agent (and, consequently, the lowest O₂ concentration) have thehighest fluorescence intensity, thus demonstrating the relationshipbetween O₂ concentration and fluorescence.

EXAMPLE 3 Use of Indicator System to Determine the Presence of aMicroorganism

[0085] A 0.5 McFarland suspension of E. coli (ATCC #25922), containingabout 1.5×10⁸ CFU/ml, was prepared using an A-Just nephelometer (AbbottLabs, Chicago, Ill.). The suspension was diluted to about 1×10⁷ CFU/mlin Broth A (see Example 1). A 150 microliter aliquot of this suspensionwas placed into indicator tray wells prepared as in Example 1, andsubsequently incubated at 37° C. At intervals, the fluorescence wasmeasured in a Fluoroskan II fluorometer over the period of 1-3½ hours.An increased fluorescence signal was observed over time as shown inFIG. 1. The fluorescence signal from wells containing no organismsshowed very little change. The wells containing organisms weresignificantly brighter when visually observed under a UV light source.Thus, it appears that the metabolic activity of the organisms in thewells caused the fluorescence signal to increase (presumably bydecreasing the O₂ concentration).

EXAMPLE 4 Dependence of Fluorescence Change on Organism Concentration

[0086] A 0.5 McFarland suspension of E. coli (ATCC #29522) in steriletrypticase soy broth (TSB, BBL #11768) was made using an A-Justnephelometer (Abbott Labs, Chicago, Ill.). A series of E. colisuspensions ranging from 1×10⁷ CFU/ml to about 10 CFR/ml were made bymaking serial dilutions. A 200 microliter aliquot of each suspension wasplaced into 8 wells of an indicator tray prepared as in Example 1. Thetray was then incubated at 37° C. and the fluorescence measured every 30minutes in a Fluoroskan II fluorometer. The fluorescence of the 8 wellswere averaged and corrected by subtracting the background fluorescenceof a sterile TSB well. The change in fluorescence over time is shown inFIG. 2.

[0087] As shown, a change in the starting concentration of the organismby a factor of 10 (one log unit) caused a delay of about 1 hour for thefluorescence in the well to exceed 2000 fluorescence units. It ispostulated that this delay is due in part to the fact that the system isopen to the atmosphere. Oxygen in the air can and does freely diffuseinto the medium in an attempt to replace that consumed by themicroorganisms. It is further postulated that only when the organismsare present in or have multiplied to sufficient numbers and aremetabolically active enough to consume oxygen at a rate approximating orfaster than the rate at which oxygen diffuses into the test solution,will the fluorescent signal generated by the indicator element in thebottom of the reservoir show an increase.

EXAMPLE 5 Preparation of an Indicator Microtitration Tray with anAlternate Fluorescent Indicating Molecule

[0088] A 96 well Microtiter tray was produced essentially as in Example1, except that tris-(2,2′ bipyridyl)-ruthenium (II) chloride hexahydrate(Aldrich Chemical Company, Milwaukee, Wis.) [Ru(BiPy)₃Cl₂] wassubstituted for Ru(DPP)₃Cl₂ in the silicone mixture. A second traycontaining 9,10-diphenyl anthracene (DPA) was also prepared. All wellswere charged with 150 ul of 1×107 CFU/ml E. coli (ATCC #25922) in broth.Table 3 lists the results at 0, 1, 2, 3, and 4 hours after addition oforganisms. TABLE 3 Fluorescence Counts for Devices with DifferentFluorophores Fluorescent Compound Silicone 0 hr. 1 hr. 2 hr. 3 hr. 4 hr.Ru(DPP)₃Cl₂ (Ex. 3) A 2300 2315 2560 8329 9000 Ru(BiPy)₃Cl₂ (Ex. 5) A2866 — 3449 3951 4109 DPA (Ex. 5) A 1300 — 1385 1456 1572 Ru(DPP)₃Cl₂(Ex. 6) B —  995 4334 3775 3508

[0089] As shown, both fluorescent sensor compounds exhibited largeincreases with fluorescence over time, indicating their suitability foruse in this system.

EXAMPLE 6 Preparation of an Indicator Microtitration Tray Using anAlternative Silicone

[0090] To demonstrate that the fluorophore can function when embedded ina different matrix, a 96 well Microtiter tray was produced essentiallyas in Example 1. In this experiment, 10 ul of white SWS-960 RTV silicone(Wacker Silicones, Adrian, Mich.) containing 10 milligrams ofRu(DPP)₃Cl₂ per liter was dispensed into each well of the tray andallowed to cure. No wash steps were performed on the resultant tray. Theresults are presented in Table 3. As in Example 1, wells containing 150ul of 1×10⁷ CFU/ml E. coli (ATCC #25922) in broth had a much greaterfluorescent intensity after several hours at 37° Centigrade.

EXAMPLE 7 Effect of Toxic Substances on the Oxygen Consumption ofMicroorganisms

[0091] A suspension containing about 3×10⁸ CFU/ml, of Pseudomonasaeruginosa (ATCC #10145) in Broth A was prepared using an A-Justnephelometer. A total of 150 ul of the suspension was placed in eachwell of the indicator trays prepared as in Example 1; these suspensionswere then diluted with solutions of phenol or copper sulfate (which aredeleterious to microbial growth) to a final concentration of 1.5×10⁸CFU/ml. The trays were incubated at 37° C. and their fluorescencemeasured in a Fluoroskan II at 10 minute intervals. FIGS. 3 and 4 showthe effect of phenol and copper sulfate on the response of the system.

[0092] As shown, at high levels of additives, growth was suppressed andthe fluorescence did not increase with time. Wells containing phenol at1 gram/liter or more, and copper sulfate at greater than 500 mg/liter,had no increase in fluorescence signal at times less than two hours,indicating absence of actively metabolizing organisms. Thus, measurementof oxygen consumption can be used to probe the metabolism of theorganisms.

EXAMPLE 8 Effect of Antibiotics on E. coli

[0093] A 0.5 McFarland suspension of E. coli (ATCC #25922) in Broth A(see Example 1) was prepared using an A-Just nephelometer. Thesuspension was diluted to 1×10⁷ CFU/ml in wells of an indicator trayprepared as in Example 1 containing the antibiotics ciprofloxacin,cefoxitin and cefuroxime at final concentrations of 0.5 to 8 ug/ml. Thetrays were incubated at 37° C. for 4 hours and their fluorescencemeasured in a Fluoroskan II fluorometer. The results are presented inTable 4. TABLE 4 Fluorescence from an Indicator Tray Containing E. coiland Antibiotics Relative Fluorescence at 4 hrs. Antibiotic Concentration(ug/mL) Ciprofloxacin Cefuroxime Cefoxitin 0.5 2537 7902 8181 1 26217983 8270 2 2461 7161 7120 4 2527 7598 3692 8 2424 6469 2974

[0094] As shown at all concentrations, the E. coli was sensitive tociprofloxacin and low fluorescence counts were observed. The E. coli wasresistant to the concentrations of cefuroxime and high fluorescencecounts were observed. The E. coli was resistant to the 0.5, 1, and 2ug/ml concentrations of cefoxitin and high counts were observed, but itwas sensitive to the higher concentrations of cefoxitin and low countswere observed for 4 and 8 ug/ml. Thus, there is a correlation betweenthe fluorescence and antibiotic concentration, demonstrating that thesystem of this invention can be used to assess the effects ofantimicrobics and to determine the minimum effective concentrationcompositions.

EXAMPLE 9 Effect of Antibiotics on the Oxygen Consumption of E. coliwith Ru(BiPy)₃Cl₂ Fluorescence Indicator

[0095] A 0.5 McFarland suspension of E. coli (ATCC #25922) in Broth A(see Example 1) was prepared using an A-Just nephelometer. Thesuspension was diluted to 1×10⁷ CFU/ml in wells of an indicator trayprepared as in Example 5 (Ru(BiPy)₃Cl₂ indicator) containing theantibiotics ciprofloxacin, cefoxitin and cefuroxime at finalconcentrations of 0.5 to 8 ug/ml. The trays were incubated at 37° C. for4 hours and their fluorescence measured in a Fluoroskan II fluorometer.The results are listed in Table 5. TABLE 5 Fluorescence from anIndicator Tray Containing E. coil and Antibiotics Relative Fluorescenceat 4 hrs. Antibiotic Concentration (ug/mL) Ciprofloxacin CefuroximeCefoxitin 0.5 507 1155 1171 1 428 1539 1491 2 308 1183 1338 4 403 1170832 8 323 1194 559

[0096] As shown, as in Example 8, at these concentrations the E. coli issensitive to ciprofloxacin and low fluorescence counts were observed.The E. coli is resistant to these concentrations of cefuroxime and highfluorescence counts were observed. The E. coli is resistant to the 0.5,1, and 2 ug/ml concentrations of cefoxitin, high counts were observed;it was sensitive to higher concentrations of cefoxitin and lower countswere observed for 4 and 8 ug/ml. Thus, the results indicated thatRu(BiPy)₃Cl₂ can also be used in a fluorescence indicator.

EXAMPLE 10 Effect of Antibiotics on the Oxygen Consumption ofMicroorganisms Using DPA Fluorescence Indicator

[0097] A 0.5 McFarland suspension of E. coli (ATCC #25922) in Broth Awas prepared using an A-Just nephelometer. The suspension was diluted to1×10⁷ CFU/ml in wells of an indicator tray prepared as in Example 5 (DPAindicator) containing the antibiotics ciprofloxacin, cefoxitin andcefuroxime at final concentrations of 0.5 to 8 ug/ml. The trays wereincubated at 37° C. for 4 hours and their fluorescence measured in aFluoroskan II. The results are presented in Table 6. TABLE 6Fluorescence from an Indicator Tray Containing E. coil and AntibioticsRelative Fluorescence at 4 hrs. Antibiotic Concentration (ug/mL)Ciprofloxacin Cefuroxime Cefoxitin 0.5 91 183 192 1 109 197 173 2 94 195164 4 74 160 101 8 68 161 95

[0098] As shown, at these concentrations the E. coli is sensitive tociprofloxacin and low fluorescence counts were observed. The E. coli isresistant to these concentrations of cefuroxime and high fluorescencecounts were observed. The E. coli is resistant to the 0.5, 1, 2 ug/mlconcentration of cefoxitin, high counts were observed; it was sensitiveto higher concentrations and lower counts were observed for 4 and 8ug/ml as in Examples 8 and 9, indicating that DPA is also useful as afluorescence indicator.

EXAMPLE 11 Effect of Open and Closed Systems on Oxygen Measurements

[0099] A 96 well indicator microtiter tray was produced substantially asin Example 1. Duplicate wells in the tray were supplemented with theantibiotic cefuroxime in the concentration range of 0.25 to 32 ug/ml.One hundred and fifty microliters of a suspension of E. coli (ATCC#11775) was added to the wells to yield about 3×10⁷ CFU/ml. One of eachduplicate well was overlaid with mineral oil to inhibit diffusion ofoxygen into the wells, the other duplicate was left open to the air. Thetray was incubated at 37° C. for 5 hours, the fluorescence was measuredin a Fluoroskan II fluorometer and that fluorescence was compared withthe average of several wells containing no antibiotic to yield a percentof the growth control at each antibiotic concentration. FIG. 5A showsthe behavior of the open and covered wells at five hours as a functionof cefuroxime concentration. FIG. 5B shows the change in fluorescence ofthe growth control wells when open or overlaid with mineral oil.

[0100] The “closed system” overlaid with mineral oil did not show aneffect on oxygen consumption by the 4 and 8 ug/ml concentrations ofantibiotic while those wells with no mineral oil showed correctly thatthis organism is sensitive to cefuroxime at these concentrations. Thisdifference is due, presumably to the time lag needed for the antibioticto affect the organism; it is believed that during this time the oxygenis brought to an artificially low level by the ongoing metabolicactivity of the organisms.

[0101] Thus, to utilize the invention with optimum sensitivity for thedetection of the effect of toxins on organisms, the sample reservoirpermits the influx of oxygen.

EXAMPLE 12 The Effect of Sample Volume on Indicator Trays

[0102] A 0.5 McFarland suspension of E. coli (BDMS Culture collection#7133) was diluted to 1×10⁷ CFU/ml in Broth A. Different volumes (from10 ul to 300 ul) of the diluted suspension were placed into wells of anindicator tray produced as in Example 1. The tray was incubated at 37°C. and the fluorescence measured in a Fluoroskan II at 30 minuteintervals. Fluorescence from the same volume of sterile broth wassubtracted to give the fluorescence change cause by the microorganism.The results are presented in Table 7. TABLE 7 Effect of Sample Volume onIndicator Tray Fluorescence Relative Fluorescence Sample Volume (ul) 0hr. 1 hr. 2 hr. 2.5 hr. 3 hr. 3.5 hr. 4 hr. 10 0 0 0 0 0 4 139 20 0 0275 795 814 1218 1958 40 0 245 683 1883 2108 2613 3240 60 0 80 1559 34974847 6226 6827 80 0 82 1798 5340 8333 8810 8801 100 0 31 1848 5952 76727962 7961 125 0 103 2798 6286 7580 7852 7852 150 0 32 2539 6005 65686759 6886 175 0 51 2574 6149 6993 6987 6798 200 0 59 2376 5355 5742 59445826 250 0 115 2172 5373 5695 5822 5759 300 0 107 2538 4650 4727 48254778

[0103] Briefly, it was observed that those wells with 40 ul or less ofsample showed less that {fraction (1/2)} the increase in relative signalobserved in wells with 80 ul or more at times of 2 hours or more. It isbelieved that in the wells containing 40 ul or less, too little volumewas present for the organisms to effectively consume oxygen faster thanit could diffuse into the small volumes of sample.

EXAMPLE 13 Use of Indicator System Without a Fluorometer

[0104] Indicator trays were prepared using the same fluorescent compoundand silicone as in Example 1. However, the trays were made of clearplastic and the wells had round bottoms (#4-3918-2, BD Labware, LincolnPark, N.J.). Two nanograms of Ru(DPP)₃Cl₂ in 10 ul of silicone wereplaced in each well of the tray and no wash steps were performed.Samples of Ps. aeruginosa (BDMS Culture collection #N111) and E. coli(ATCC #25922) were diluted to Broth A (see Example 1) 1×10⁷ CFU/ml inBroth A containing either 0 to 32 ug/ml cefuroxime, 0.12 to 8 ug/mlciprofloxacin or 0 to 32 ug/ml cefoxitin and charged to the trays. Thetrays were incubated for 4 hours at 37° C. and subsequently placed onthe stage of an ultraviolet transilluminator (#TX-365A, SpectronicsCorp., Westbury, N.Y.) which served as an excitation source. Theresulting fluorescence was observed from directly above the trays at adistance of 1 foot through a 550 cut-on filter (#LL-550 -S-K962, Corion,Holliston, Mass.). It was readily observed that wells which containedeither no antibiotics or concentrations of antibiotics that did notaffect the organisms demonstrated a high level of fluorescence. Wellswith either no organisms or higher antibiotic levels had a much lowerlevel of fluorescence. The lowest concentration of antibiotic tosignificantly lower the fluorescent emissions for each organism is shownin Table 8 along with the MIC concentration determined using anovernight microdilution antimicrobial susceptibility test. TABLE 8Fluorescence Results Obtained Without Use of an Instrument MICCefuroxime Ciprofloxacin Cefoxitin Visual Reference Visual ReferenceVisual Reference Ps. aeruginosa >64 >64 1 0.5 >64 >64 E. coli #25922 168 <0.12 <0.12 8 4

EXAMPLE 14 Use of Indicator to Detect the Presence of a Low Level ofBacteria In a Medium Containing Blood

[0105] Tissue culture flasks (Falcon #3084, BD Labware, Lincoln ParkN.J.) were prepared with one side coated with 3 mls of Dow CorningWater-based Emulsion containing 68 ng of Ru(DPP)₃Cl₂. The flasks weresterilized using ethylene oxide. One hundred thirty five mls of TSBbroth (BBL #11768) containing about 0.05 CFU/ml E. coli (ATCC #25922)and 15 mls of defibrinated sheep blood was added to one of the flasks. Acontrol flask contained 135 mls of TSB and 15 mls of blood but noorganisms. The caps of the flasks were loosened to allow air circulationand the flasks were incubated at 37° C. in an upright position. A fiberoptic probe allowed the fluorescence from the flasks to be measured by aPerkin Elmer LS-5B spectrofluorometer located several feet from theincubator. The fluorometer measured the flasks at 485 nm excitationwavelength with a 10 nm slit width and a 550 nm cut-on emission filter.A strip chart was attached to the fluorometer and the fluorescencemonitored continuously for 16 hours. At 7.5, 10.5 and 16 hours duringthe incubation period a 100 ul aliquot was removed from the test flask,diluted 1:100 in sterile TSB and 100 ul of the dilution was spread oneach of three TSA plates to determine the number of CFU/ml present inthe flask. The results are graphically depicted in FIG. 6.

[0106] As shown, the non-invasive techniques of this invention can beused for the detection of organisms in blood, a very critical anddemanding task. The flask contained a very cloudy and turbid solutionwhich is continuously monitored for sixteen hours, and measurement offluorescence showed a direct correlation to the growth of organisms.This growth was readily detected by 11 hours, when the concentration oforganisms had just exceeded 10⁶ CFU/ml.

EXAMPLE 15 Indicator Coated on the Spherical Ends of FAST Tray LidProngs

[0107] This example monitored bacterial respiration with oxygenindicators coated on the spherical ends of FAST tray (Becton Dickinson)lid prongs. Three different indicators were evaluated.

[0108] The first indicator prepared was a mixture of 1 ml of 2 mg/mldichloromethane solution of Ru(DPP)₃Cl₂ and 10 ml Dow-Corning 3-5024water-based silicone emulsion. The spherical ends of FAST tray lidprongs were dipped into a shallow reservoir of the indicator solution,removed, placed prong side down in a rack, and allowed to cure byevaporation. The second indicator was prepared by mixing 3mL WackerSWS-960 clear silicone dispersion, 6 mL petroleum ether, and 0.5 mL ofthe 2 mg/mL dichloromethane solution of Ru(DPP)₃Cl₂. The spherical endsof FAST tray lid prongs were coated with this indicator in the samemanner as with the first indicator and allowed to cure by evaporation ofthe solvents and reaction with atmospheric moisture. The third indicatorwas prepared in the same manner as the second but Wacker SWS-960 whitesilicone was used.

[0109] A 1×10⁷ CFU/mL suspension of E. coli ATCC #25922 in MuellerHinton broth was prepared; 150 microliter aliquots were pipetted intothe odd numbered rows of a microtiter tray, while 150 microliteraliquots of uninoculated Mueller Hinton broth were pipetted into thewells of the even numbered columns. The lids containing the indicatorcoated prongs were placed on the trays. The lidded trays were placed ina 37° C. high humidity incubator for 3 hours.

[0110] Following the three hour incubation, the trays were placed on atransparent glass plate. A mirror was positioned below the glass platein such a manner that the bottom of the tray was visible in the mirror.A 365 nm ultraviolet source which evenly illuminated the entire tray waspositioned about one inch from the top of the tray. A box, with a smallwindow through which the mirror could be seen, was placed over theassembly to block room light, and a 550 nm cut-on filter was placed inthe box window. With this assembly the fluorescence from the indicatorcoated spherical ends of the FAST tray lid prongs could be visualizedthrough the tray bottom. Table 9 contains the results of visualobservations of the trays evaluated in this manner. TABLE 9 VisualObservations of Indicator Coated Lid Prongs Viewed Through Tray BottomsSilicone Observations Dow-Corning Very bright fluorescence from spheresimmersed in organism containing wells. Very weak fluorescence fromprongs in uninoculated wells. Wacker Clear Some visible differencebetween prongs immersed in inoculated and uninoculated wells. Differencemuch less observable than with Dow- Corning indicator. Wacker White Verybright fluorescence from spheres immersed in inoculated wells, intensityabout equal to Dow- Corning indicator. Some weak fluorescence fromspheres in uninoculated wells.

[0111] Thus, all three indicator systems produced desirable results,with the Dow Corning and Wacker White exhibiting much moredistinguishable differences between the inoculated and uninoculatedwells.

EXAMPLE 16 Indicators Consisting of Ru(DPP)₃Cl₂ Adsorbed on Silica GelParticles Embedded in UV Cured Silicone Rubber

[0112] Indicators were prepared by adsorbing Ru(DPP)₃Cl₂ onto silica gelparticles and embedding these particles into Loctite Nuva-Sil siliconerubbers. A variety of indicators were prepared using silica gelparticles of different mesh sizes, different amounts of adsorbedfluorophore, different ratios of silica gel to silicone, and two typesof Loctite Nuva-Sil (Nuva-Sil 5091 and Nuva-Sil 5147). Table 10 containsthe characteristics of the indicators prepared and the visual resultsobtained from the indicators in contact with microorganism suspensions.An exemplary procedure used for the preparation of the indicators ispresented below.

[0113] Ten grams of 100-200 mesh Davisil silica gel (Aldrich, Milwaukee,Wis.) was weighed into a 500 mL round bottom evaporation flask. Fortythree milliliters of a 0.14 mg/mL ethanol solution of Ru(DPP)₃Cl₂ waspipetted into the flask. The ethanol was removed by rotary vacuumevaporation resulting in the adsorption of the Ru(DPP)₃Cl₂ on the silicagel at a concentration of 0.6 mg Ru(DPP)₃Cl₂/gm silica gel. Four gramsof this silica gel were mixed with 16 g Loctiote Nuva-Sil 5091 (Locite,Newington, Conn.) resulting in a 20% w/w silica/silicone ratio.Twenty-five microliter aliquots of this mixture were pipetted into thewells of a microtiter tray. The silicone was cured by exposure to highintensity ultraviolet radiation for 15 seconds in a Loctite Zeta 7200 UVcuring chamber. The other indicators in Table 10 were similarlyprepared.

[0114] To evaluate the indicators, 150 microliters of a 1×10⁷ CFU/mLsuspension of E. coli (ATCC #25922 in Mueller Hinton II broth (BBL) waspipetted into selected wells of the microtiter tray; uninoculated brothwas pipetted into other wells. The tray was incubated in a high humidity35° C. incubator for 3 hours. To visualize the fluorescence from theindicator the tray was placed on the stage of a 365 nm UVtransilluminator; the fluorescence from the indicator was observed fromabove through a 550 nm cut-on filter. A “+” sign in the Response columnof Table 10 indicates that a visibly discernible increased fluorescencewas observed from the wells containing the organism. TABLE 10 IndicatorFormulations and Responses Mesh Size mg Ru(DPP)₃Cl₂/g Silica Wt % SilicaSilicone Response  60-100 0.2, 0.4, 0.6 5, 10, 20 5091, +* 5147 100-2000.2, 0.4, 0.6 5, 10, 20 5091, +* 5147 200-425 0.2, 0.4, 0.6 5, 10, 205091, +* 5147

[0115] In replicate trials utilizing wells with no microorganisms, theindicators displayed little or no light (although at higher (0.6 mg/gm)concentrations of indicator, a dim fluorescence was noted).

EXAMPLE 17 Oxygen Sensor Not In Direct Contact With Sample Fluid

[0116] Test vials (80 mL volume) containing 60 mL of media and oxygensensor (OS) were inoculated with the following organisms: Pseudomonasaeruginosa, Mycobacterium fortuitum and Escherichia coli. The vials wereconnected to 80 mL vials without broth with oxygen impermeable rubbertubing. The vials were then entered into adjacent stations in a BACTEC®9240 instrument. Data was collected on the two vials over a 50 hourperiod. The results of these tests are presented in FIGS. 7 through 9.FIG. 7 depicts the data collected in the BACTEC® instrument indicatingthe change in fluorescence intensity indicative of the growth of P.aeruginosa. FIG. 8 depicts the data collected in the BACTEC® instrumentindicating the change in fluorescence intensity indicative of the growthof M. fortuitum. FIG. 9 depicts the data collected in the BACTEC®instrument indicating the change in fluorescence intensity indicative ofthe growth of E. coli. For each of the figures the bold line in thesefigures represent the data collected in the vials containing broth; thelight lines represent the data collected by the sensor that is not indirect contact with the liquid broth. In all three cases, oxygenconsumption was observed in the vials without broth. The pattern ofoxygen consumption exhibited in these vials indicates logarithmic oxygenconsumption which is indicative of microbial growth.

[0117] The data shows that the OS was used for the detection ofmicrobial growth in the absence of direct broth to sensor contact. Thedetection delays observed in the vials without media are related to thisparticular test configuration. One having ordinary skill in this artwould be able to optimize the parameters of the system, by example andnot limitation, such as, by reducing the headspace volume and oxygenconcentration which would result in improved sensitivity and make themeasurements made without direct contact of liquid broth (gas phase)more comparable with the measurements made with contact of the liquidbroth (liquid phase).

EXAMPLES 18-26

[0118] Methods and Materials: Oxygen Sensor Plate preparation

[0119] Oxygen sensor plates were prepared by the general methodsdescribed herein. Falcon 1177 polystyrene 96 well U-bottom plates (BDLabware) were used for all experiments. The fluorescent dye Ru(DPP)₃Cl₂was adsorbed to silica gel by rotary evaporation of ethanolic solutionsof the dye with the silica gel. The adsorbed dye-silica and amoisture-cure clear silicone were mixed manually and immediately appliedto plate wells with approximately 17 uL silicone per well. These werecured. for 2-3 days in a controlled humidity incubator.

[0120] Microwell Plate Fluorescence Assays

[0121] All data was obtained with a BMG Polarstar fluorimeter at 37° C.using the bottom plate reading configuration. The bandpass filters were465 nm for excitation and 590 nm for emission. For the experiments withMATRIGEL®, a Cytofluor 4000 fluorometer was used with a 485 nmexcitation filter and a 580 nm emission filter. Data was read atselected time intervals. Normalized fluorescence data was generallyobtained by dividing well values at selected time points by the samewell's initial reading with only media or buffer present prior to addingcells.

[0122] Cells used for cytotoxicity and quantitation experiments (HL60,U937; ATCC) were grown in tissue culture media recommended by thesupplier (RPMI; Gibco and ATCC, respectively). The media wassupplemented with either 20% fetal bovine serum (Hyclone) for the HL60cells or 10% FBS for the U937 cells, with the addition of penicillin,streptomycin and fungizone (Gibco) to prevent microbial contamination.Cells were maintained in a tissue culture incubator (37° C., 5% CO₂, 95%humidity) during all experiments between readings.

EXAMPLE 18 Quantitation of Fluorescent Signal vs. Cell Number for HL60Cells Growing in Oxygen Sensor Plates

[0123] HL60 cells (human promyelocytic leukemia cell line, ATCC #45500)were grown in tissue culture media recommended by the supplier (RPMI;Gibco), supplemented with 20% fetal bovine serum, heat inactivated at50° C. for thirty minutes with the addition of penicillin, streptomycinand fungizone (Gibco) to prevent microbial contamination. Cells weremaintained in tissue culture incubator (37° C., 5% CO₂, 95% humidity)during all experiments. Fluorescence of the oxygen sensor was read on aPolarstar™ fluorometer (BMG), using 465 nm excitation and 590 nmemission filters.

[0124] 100 uL of tissue culture media was aliquoted into the wells ofthe Oxygen sensor plate and plate was allowed to equilibrate in thetissue culture incubator for 1 hour prior to taking the initial reading.This reading was used to normalize all subsequent readings to accountfor well to well variability. Cells were resuspended in fresh media at960,000 cells/ml. Serial 1:2 dilution of this stock was performed and100 uL of each dilution was alliquoted across the length of the plate inreplicates of 12 (rows B-H), with final cell/well number from 1,500 to96,000. In row A, 100 uL of tissue culture media was alliquoted into thewells in lieu of cells (no-cells control). Fluorescence measurementswere taken every 24 hours over 5 days. The actual number of cellspresent was counted with a hemocytometer by sampling the parallel well(same seeding cell number). Mean fluorescence from 5 wells per datapoint (n=5) was plotted against the counted cell number (FIG. 10). Errorbars are standard deviation of the mean.

EXAMPLE 19 Quantitation of Fluorescent Signal vs. Cell Number for U937Cells Growing in Oxygen Sensor Plates

[0125] This experiment was performed similarly to the one in Example 18.U937 cells (human histiocytic lymphoma cell line, ATCC, #CRL-1593.2)were grown as above, with the exception that 10% fetal bovine serum wasused. Cell number varied from 750 to 48,000 cells per well. Fluorescencewas measured at times indicated and plotted against seeded (initial)cell number (FIG. 11).

EXAMPLE 20 Cytotoxicity of Vinblastine Assayed By Oxygen Sensor

[0126] The experiment was performed as described in Example 18, with thefollowing exceptions. Serial dilutions of vinblastine were prepared attwice the final concentration (100 nM to 0.1 nM) in tissue culturemedia.100 uL of the drug dilution in tissue culture media was aliquotedacross the width of the plate in replicates of five, reserving onecolumn for no-drug (media only) control. After an initial reading, aconstant number of cells (200,000/well) in 100 uL media were added toeach well of rows A-E of the oxygen sensor plate, reserving rows F-H forno-cells control. Fluorescence was read at the indicated times. Meanfluorescence from 5 wells per data point (n=5) was plotted againstvinblastine concentration (FIG. 12).

EXAMPLE 21 Cytotoxicity of Vinblastine Assayed by MTT

[0127] In parallel with the oxygen sensor assays, MTT assays wereperformed using a Cell Titer Kit™ (Promega), as in Example 20, with thefollowing exceptions. For each time point (corresponding to oxygensensor assay time point), one flat-bottom 96 well microtiter plate(Falcon) was used. 50 uL of drug dilution in tissue culture media wasused for initial reading and cells were suspended in 50 uL media, to thefinal volume of 100 uL. At indicated time points, 10 uL of MTT reagentwas added per well for 1 hour, after which 100 uL of the stop/lysisbuffer was added. Plates were sealed with Parafilm™ and cell lysisoccurred overnight. Absorbance (570 nm corrected by absorbance at 750nm) was read using a Thermomax Microplate Reader (Molecular Devices).Mean absorbance from 5 wells per data point, with standard deviation aserror bars, was plotted against vinblastine concentration (FIG. 13).

EXAMPLES 22-24 Cytotoxicity of Methotrexate, Sodium Azide, and SDS(Sodium Dodecyl Sulfate) Assayed by Oxygen Sensor Plates and MTT Assays

[0128] These experiments was performed as described in Example 20, withthe exception that appropriate dilutions of the above reagents weresubstituted for vinblastine: 0.01 nM to 10000 nM methotrexate, 0.00001to 10 mM sodium azide, and 2 to 2000 uM SDS respectively. Complete doseresponse curves for these three additional drugs are shown, respectivelyin FIGS. 14, 15 and 16. TABLE 11 Comparison of IC₅₀ ** Values ForSelected Drugs With HL60 Cells Obtained With Oxygen Sensor Plates andMTT Assays Time (hours) 24 48 72 96 120 O₂ Sensor MTT O₂ Sensor MTT O₂Sensor MTT O₂ Sensor MTT O₂ Sensor MTT Vinblastine * * 11 nM  9 nM 11 nM 8 nM  9 nM 7 nM 9 nM 6.4 nM Methotrexate * * * * * * 19 nM  * 12 nM  14nM SDS 340 uM 500 uM 300 uM  440 uM 300 uM  430 uM * * * * SodiumAzide * * 13 uM  25 uM 13 uM  25 uM 9 uM 3 uM 5.8 uM  1.6 uM

[0129] Discussion (for Table 11 and Examples 20-24): Cytotoxicity assayswere performed in parallel with oxygen sensor plates and with standardMTT assays to measure the cytotoxicity of four drugs/toxins:vinblastine, methotrexate, sodium azide, and sodium dodecylsulfate. Bothassay methods gave comparable IC₅₀ values at the selected timepoints.Because the MTT assay is an endpoint assay requiring additional reagentsand destruction of the cells, it required a separate plate for eachtimepoint. The oxygen sensor assay, however, required only a singleplate for all timepoint readings with each drug, significantly reducingthe amount of labor and materials for each IC₅₀ determination over time.

[0130] Conclusion: For each timepoint in these cytotoxicity assays wheresignificant drug or toxin effect could be measured, the IC50 valuesobtained for the oxygen sensor plate matched closely with the MTTvalues.

EXAMPLE 25 Oxygen Consumption by Mammalian Cells Growing on MATRIGEL®

[0131] Various amounts (100 μl, 50 μl, 25 μl, or 0 μl) of MATRIGEL® (BDLabware cat. #4024C) were added to the wells of an ice-cold O₂ sensortray and then allowed to gel at room temperature. The plate was moved toa 37° C. incubator before use. MCD-1 cells (Moore et al. (1996) In VitroProperties of a Newly Established Meulloblastoma Cell Line MCD-1. Mol.Chem. Neuropath. 29, 107-126) were suspended by trypsinization, washedwith DMEM/F12 medium containing 10% fetal calf serum, and resuspended to5×10⁵ cells/ml in the same medium without serum. In some experiments,Hepes buffer (10 mM, pH 7.4) was added to better control the pH duringthe incubation period. Cells (100 μl, 5×10⁴ cells) were added to thewells and the plate was moved to a modified PerSeptive BiosystemsCytofluor fluorimeter at 37° C. The fluorescence was read over timeusing 485 nm excitation and 580 nm emission filters.

[0132] There was a clear increase in fluorescence signal in wellscontaining MCD-1 cells compared to wells without cells (FIG. 17). Thesignal increased more slowly in wells containing increasing amounts ofMATRIGEL®, suggesting there may be a barrier to the migration of cellstoward the silicone sensor or to the diffusion of oxygen through theMATRIGEL®. Much of the increase in signal occurred over the first fewhours of the experiment.

EXAMPLE 26 MCD-1, SK-N-SH, and NIH3T3 Cells Growing on MATRIGEL® Differin Their Rates of Oxygen Consumption

[0133] The O₂ sensor with or without 50 μl MATRIGEL® per well wasprepared as described above. MCD-1, SK-N-SH (American Tissue TypeCollection HTB11), or mouse fibroblast NIH3T3 cells (American TissueType Collection CRL1658) were collected by trypsinization, washed andadded to wells as above at 5×10³ or 5×10⁴ cells per well. The plateswere moved to a 37° C. fluorimeter and monitored as above.

[0134] For MCD-1 cells (FIG. 18A), the fluorescence signal developedmore slowly in the wells containing MATRIGEL® than in the wells lackingMATRIGEL®. However, the final levels of fluorescence were comparable.The signal with 5×10³ MCD-cells per well was much smaller than thesignal with 5×10⁴ cells, but was above the “no cells” control. Inaddition, the oxygen consumption by the MCD-1 cells was inhibited by0.1% sodium azide, as also observed for suspension cell cultures. Thesignal with 5×10⁵ SK-N-SH cells (FIG. 18B) was very clearly abovebackground and moreover, appeared to show a second increase beginning atabout 6 hr, perhaps due to cell division. A third cell line, 3T3, showedan oxygen consumption between that of MCD-1 and SK-N-SH cells (FIG.18C). Although the three cell lines differ markedly in their metabolicrates, oxygen consumption could be detected in all cases by a properchoice of cell number.

[0135] Table 12 presents a summary for some of the mammalian cell typesand corresponding conditions for growth which have been used with oxygensensor plates. TABLE 12 Cell lines which have been tested in OxygenSensor Plates Cell Line Cell type Media Contains Supplements AdherentCells SK-N-SH Human S-MEM Pen/Strep/Fungizone neuroblastoma Gibco EaglesSalts Nonessential Amino Acids cat# 11380 L-glutamine Sodium PyruvateContains 10% Fetal Bovine Serum MCD-1 Human D-MEM F12 15 mM HepesPen/Strep/Fungizone Medulloblastoma Gibco L-glutamine 10% Fetal BovineSerum cat# 11330 pyrodoxine HCl WI-38 Human MEM Earles SaltsPen/Strep/Fungizone Fibroblast Gibco L-glutamine Nonessential AminoAcids embroyonic lung cat # 11095 Sodium Pyruvate 10% Fetal Bovine SerumNIH-3T3 Mouse D-MEM F12 15 mM Hepes No supplements Fibroblast GibcoL-glutamine cat# 11330 pyrodoxine HCl Nonadherent Cells HL60 Human RPMIL-glutamine Pen/Strep/2X Fungizone promyelocytic Gibco 20% Fetal BovineSerum, leukemia cat # 11875 heat inactivated U-937 Human RPMIL-glutamine Pen/Strep/2X Fungizone histiocytic lymphoma Gibco 10% FetalBovine Serum cat # 11875 ATCC 10 mM Hepes Pen/Strep/2X Fungizone cat#30-2001 1 mM Na pyruvate 10% Fetal Bovine Serum 4 g/L glucose 1.5 g/Lbicarbonate 2 mM glutamine

EXAMPLE 27 Oxygen Sensor Added to Bottom of Well Insert Membrane

[0136] This example demonstrates an alternate format for using thesensors to monitor cells and provides a method for monitoring the growthof an adherent cell line (MDCK) by applying the sensor to the exteriorof a cell culture insert membrane which supports growth of adherentcells.

[0137] The oxygen sensor silicone formulation was prepared by thegeneral methods described herein and 50 uL was applied to one half ofthe wells of a 24 well microplate (Falcon product 3047). In addition, 20uL of the sensor silicone mixture was added to the exterior side of thetrack-etched PET (polyethylene terephthalate) membrane of 24-well plateinserts (Falcon 3097). These sensors were cured at 37° C. for 2 days.

[0138] Cells in these modified inserts were monitored in the unmodifiedwells of the 24 well plate and compared to corresponding cell linesgrown in modified wells with the sensor on the well bottom but withoutan insert present. Cells were grown and monitored by fluorescence as inthe preceding examples. Briefly, 300,000 HL60 cells were added to eachmodified insert or well and 100,000 MDCK cells were added to each insertor well. The cells were added in the corresponding media for each celltype (see Table 12): 0.7 mL per well and 0.3 mL per insert. Controlwells with media only and no cells were also monitored. Readings weretaken daily for nine days.

[0139] The results are shown in FIGS. 19A and 19B. For both the adherentcells and non-adherent cells a greater signal was obtained more quicklywith the modified insert sensors.

EXAMPLE 28 Preparation of a 384 Well Sensor Plate

[0140] The general methods described above and in Example 18 were usedto prepare a 384 well sensor plate (Nunc #242765 clear polystyrene) with10 uL of sensor per well. A titration of HL60 cells in 100 uL media wasmonitored with the BMG fluorescence plate reader.

[0141] The results are shown in FIG. 20 and compared to thecorresponding 96 well plate results. The 384 well plate demonstrated animproved time-to-signal response for the same number of HL60 cells perwell.

EXAMPLE 29 Detection of SF-9 Insect Cell Growth

[0142] SF-9 insect cells are derived from the pupal ovarian tissue ofthe fall army worm, Spodoptera frugiperda (D. R. O'Reilly, et al (1992)The Baculovirus System: A Laboratory Guide, Chapman and Hall, NYC,N.Y.). A 96 well oxygen sensor plate was equilibrated for 1 hour at 27°C. with 100 μL TMN-FH media (Invitrogen, Inc., Graces Insect mediasupplemented with 10% fetal calf serum, and powdered form of yeastolate,lactalbumin hydrolysate and glutamine).

[0143] Seven concentrations of serially diluted SF-9 cells were added inreplicates of five across the O₂ sensor plate starting at 160,000cells/well (800,000 cells/mL) down to 1,500 cells/well (7,500 cells/mL).Cells were incubated at 27° C. in a humidity chamber and fluorescencewas monitored over time using the same instrument and parameters used inthe mammalian cell experiments.

[0144] The signal increased more rapidly and to a greater degree thanmammalian cells (FIG. 21). The wells with 160,000 cells/well reachedmaximum fluorescence within 2 hours. As with other cellular experiments,the initial cell number in a well can be estimated by the time requiredto reach a measurable fluorescence increase per well.

EXAMPLE 30 Monitoring Yeast Growth

[0145] Growth of a sample of Saccharomyces cerevisiae was monitored in a96 well oxygen sensor plate (prepared as described in Example 16). Aninitial yeast broth was prepared by hydrating 11.5 g of dried Edme AleYeast (Edme, Ltd., UK) in 100 mL water at 37° C. for 30 minutes. Thisyeast slurry was added to a 500 mL mixture of NZCYM Media (BBL #99165)plus 10 g/L d-glucose. After 36 hours of fermentation at 27° C. (justpast exponential growth phase) the yeast cell concentration wasdetermined using a hemacytometer. Serial dilutions were prepared from1.2×10⁷ cells/mL down to 1.6×10⁴ cells/mL in fresh NZCYM broth witheither 10, 50, 100, or 200 g/L d-glucose. Each suspension was measuredin triplicate (200 uL /well) in a 96 well sensor plate at 27° C. withcontinuous readings every 400 sec for 13.3 hours.

[0146]FIG. 22 shows the fluorescence signal for the yeast cell titrationin 10 g/L glucose. This indicates the relationship between initialconcentration and time required to generate a positive signal. FIG. 23compares yeast growth (as determined by time required to reach 120% ofinitial fluorescence) vs. glucose concentration. The two lower glucoseconcentrations (10 and 50 g/L) indicate a more linear relationship tothe initial yeast concentration, whereas the two higher glucoseconcentrations (100 and 200 g/L) appear to retard initial growth of lowyeast concentrations. This demonstrates one way the sensor plates can beused for optimizing cellular growth conditions.

[0147] It is apparent that many modifications and variations of thisinvention as hereinabove set forth may be made without departing fromthe spirit and scope of the present invention and the above examples arenot intended to in any way limit the present invention but are merelyexemplary.

What is claimed is:
 1. A method for determining the presence or absenceof respiring eukaryotic cells in a solution comprising: (i) contactingsaid solution with a sensor composition which comprises a luminescentcompound that exhibits a change in luminescent property, when irradiatedwith light containing wavelengths which cause said compound toluminesce, upon exposure to oxygen, wherein the presence of the sensorcomposition is nondestructive to the eukaryotic cells; (ii) irradiatingsaid sensor composition with light containing wavelengths which causesaid luminescent compound to luminesce; (iii) measuring or visuallyobserving the luminescent light intensity from said luminescent compoundwhile irradiating said sensor compound with said light; (iv) comparingsaid measurement to that of a control not containing respiringeukaryotic cells, wherein said control is selected from the groupconsisting of: a reagent control not in contact with respiringeukaryotic cells and a calculated threshold, wherein a change inluminescent property relative to the luminescent property of the controlis indicative of the presence of respiring eukaryotic cells; and (v) inthe event that no such increase is measured or observed, repeat steps(ii), (iii), and (iv) as needed, to determine the presence or absence ofrespiring eukaryotic cells in said solution.
 2. The method of claim 1wherein said luminescent compound is contained within a matrix which isrelatively impermeable to water and non-gaseous solutes, but which has ahigh permeability to oxygen.
 3. The method of claim 2 wherein saidmatrix is a rubber or plastic matrix.
 4. The method of claim 2 whereinsaid matrix is a silicone rubber matrix.
 5. The method of claim 2wherein said luminescent compound is adsorbed on solid silica particles.6. The method of claim 1 wherein said luminescent compound is atris-4,7-diphenyl-1, 10-phenanthroline ruthenium (II) salt.
 7. Themethod of claim 6 wherein said luminescent compound is tris-4,7-diphenyl-1, 10-phenanthroline ruthenium (II) chloride.
 8. The methodof claim 1 wherein said luminescent compound is a tris-2, 2′-bipyridylruthenium (II) salt.
 9. The method of claim 8 wherein said luminescentcompound is tris-2, 2′-bipyridyl ruthenium (II) chloride hexahydrate.10. The method of claim 1 wherein said luminescent compound is 9,10-diphenyl anthracene.
 11. The method of claim 1 wherein said solutionis isolated from atmospheric oxygen wherein said solution is containedin a closed system.
 12. The method of claim 1 wherein said solution isexposed to atmospheric oxygen.
 13. The method of claim 1 wherein, instep (i), the solution is also contacted with an effective concentrationof one or more biomaterials.
 14. The method of claim 13 wherein saidbiomaterial is MATRIGEL®.
 15. The method of claim 1 wherein, in step(i), the solution is also contacted with an effective concentration ofone or more extracellular matrices.
 16. The method of claim 15 whereinsaid extracellular matrix is collagen.
 17. The method of claim 1wherein, in step (i), the solution is contacted with an effectiveconcentration of one or more additives or coating substances.
 18. Amethod for determining the effects of at least one drug, toxin orchemical on respiring eukaryotic cells comprising: (i) preparing aliquid media broth of said eukaryotic cells; (ii) contacting said brothwith a sensor composition which comprises a luminescent compound thatexhibits a change in luminescent property, when irradiated with lightcontaining wavelengths which cause said compound to luminesce, uponexposure to oxygen, wherein the presence of the sensor composition isnon-destructive to the eukaryotic cells; (iii) admixing with said brotha quantity of said drug, toxin or chemical; (iv) irradiating said sensorcomposition with light containing wavelengths which cause saidluminescent compound to luminesce; (v) measuring or visually observingthe change in luminescent property from said luminescent compound whileirradiating said sensor compound with said light; and (vi) comparingsaid measurement to that of a control wherein said control is selectedfrom the group consisting of: a reagent control not in contact withrespiring eukaryotic cells or the drug, toxin or chemical; a reagentcontrol in contact with respiring eukaryotic cells but not in contactwith the drug, toxin or chemical and a calculated threshold, wherein achange in luminescent property relative to the control is indicative ofcytotoxicity of the drug, toxin or chemical to the eukaryotic cells; and(vii) in the event that no such change is measured or observed, repeatsteps (iv), (v) and (vi), as needed, to determine the effects of thedrug, toxin or chemical on the respiring eukaryotic cells.
 19. Themethod of claim 18 wherein said luminescent compound is contained withina matrix which is relatively impermeable to water and non-gaseoussolutes, but which has a high permeability to oxygen.
 20. The method ofclaim 19 wherein said matrix is a rubber or plastic matrix.
 21. Themethod of claim 19 wherein said matrix is a silicone rubber matrix. 22.The method of claim 19 wherein said luminescent compound is adsorbed onsolid silica particles.
 23. The method of claim 18 wherein saidluminescent compound is a tris-4, 7-diphenyl-1, 10-phenanthrolineruthenium (II) salt.
 24. The method of claim 23 wherein said luminescentcompound is tris4, 7-diphenyl-1, 10-phenanthroline ruthenium (II)chloride.
 25. The method of claim 18 wherein said luminescent compoundis a tris-2, 2′-bipyridyl ruthenium (II) salt.
 26. The method of claim25 wherein said luminescent compound is tris-2, 2′-bipyridyl ruthenium(II) chloride hexahydrate.
 27. The method of claim 18 wherein saidluminescent compound is 9, 10-diphenyl anthracene.
 28. The method ofclaim 18 wherein said broth is isolated from atmospheric oxygen.
 29. Themethod of claim 18 wherein said broth is exposed to atmospheric oxygen.30. The method of claim 18 wherein, in step (ii), the liquid media isalso contacted with an effective concentration of one or morebiomaterials.
 31. The method of claim 30 wherein said biomaterial isMATRIGEL®.
 32. The method of claim 18 wherein, in step (ii), the liquidmedia is also contacted with an effective concentration of one or moreextracellular matrices.
 33. The method of claim 32 wherein saidextracellular matrix is collagen.
 34. The method of claim 18 wherein, instep (ii), the liquid media is contacted with an effective concentrationof one or more additives or coating substances.
 35. A method forquantifying respiring eukaryotic cells in a solution comprising: (i)contacting said solution with a sensor composition which comprises aluminescent compound that exhibits a change in luminescent property,when irradiated with light containing wavelengths which cause saidcompound to luminesce, upon exposure to oxygen, wherein the presence ofthe sensor composition is non-destructive to the eukaryotic cells; (ii)irradiating said sensor composition with light containing wavelengthswhich cause said luminescent compound to luminesce; (iii) measuring orvisually observing the change in luminescent property from saidluminescent compound while irradiating said sensor compound with saidlight; (iv) comparing said measurement to that of a control notcontaining respiring eukaryotic cells, wherein said control is selectedfrom the group consisting of: a reagent control not in contact withrespiring eukaryotic cells and a calculated threshold, wherein a changein luminescent property relative to the luminescent property of thecontrol is indicative of the presence of respiring eukaryotic cells; and(v) in the event that no such increase is measured or observed, repeatsteps (ii), (iii), and (iv) as needed, to quantify respiring eukaryoticcells in said solution.
 36. The method of claim 35 wherein saidluminescent compound is contained within a matrix which is relativelyimpermeable to water and nongaseous solutes, but which has a highpermeability to oxygen.
 37. The method of claim 36 wherein said matrixis a rubber or plastic matrix.
 38. The method of claim 36 wherein saidmatrix is a silicone rubber matrix.
 39. The method of claim 36 whereinsaid luminescent compound is adsorbed on solid silica particles.
 40. Themethod of claim 35 wherein said luminescent compound is a tris-4,7-diphenyl-1, 10-phenanthroline ruthenium (II) salt.
 41. The method ofclaim 40 wherein said luminescent compound is tris-4, 7-diphenyl-1,10-phenanthroline ruthenium (II) chloride.
 42. The method of claim 35wherein said luminescent compound is a tris-2, 2′-bipyridyl ruthenium(II) salt.
 43. The method of claim 42 wherein said luminescent compoundis tris-2, 2′-bipyridyl ruthenium (II) chloride hexahydrate.
 44. Themethod of claim 35 wherein said luminescent compound is 9, 10-diphenylanthracene.
 45. The method of claim 35 wherein said solution is isolatedfrom atmospheric oxygen wherein said solution is contained in a closedsystem.
 46. The method of claim 35 wherein said solution is exposed toatmospheric oxygen.
 47. The method of claim 35 wherein, in step (i), thesolution is also contacted with an effective concentration of one ormore biomaterials.
 48. The method of claim 47 wherein said biomaterialis MATRIGEL®.
 49. The method of claim 35 wherein, in step (i), thesolution is also contacted with an effective concentration of one ormore extracellular matrices.
 50. The method of claim 49 wherein saidextracellular matrix is collagen.
 51. The method of claim 35 wherein, instep (i), the solution is contacted with an effective concentration ofone or more additives or coating substances.
 52. A method fordetermining the presence or absence of respiring eukaryotic cells in asolution comprising: (i) placing said solution in a container in whichsaid fluid is substantially isolated from atmospheric oxygen and placingwithin said container, but not in direct contact with said fluid, asensor composition which comprises a luminescent compound that exhibitsa change in luminescent property, when irradiated with light containingwavelengths which cause said compound to luminesce, upon exposure tooxygen, wherein the presence of the sensor composition isnon-destructive to the eukaryotic cells; (ii) irradiating said sensorcomposition with light containing wavelengths which cause saidluminescent compound to luminesce; (iii) measuring or visually observingthe luminescent light intensity from said luminescent compound whileirradiating said sensor compound with said light; (iv) comparing saidmeasurement to that of a control not containing respiring eukaryoticcells, wherein said control is selected from the group consisting of: areagent control not in contact with respiring eukaryotic cells and acalculated threshold, wherein a change in luminescent property relativeto the luminescent property of the control is indicative of the presenceof respiring eukaryotic cells; and (v) in the event that no suchincrease is measured or observed, repeat steps (ii), (iii), and (iv) asneeded, to determine the presence or absence of respiring eukaryoticcells in said solution.
 53. The method of claim 52 wherein saidluminescent compound is contained within a matrix which is relativelyimpermeable to water and non-gaseous solutes, but which has a highpermeability to oxygen.
 54. The method of claim 53 wherein said matrixis a rubber or plastic matrix.
 55. The method of claim 53 wherein saidmatrix is a silicone rubber matrix.
 56. The method of claim 53 whereinsaid luminescent compound is adsorbed on solid silica particles.
 57. Themethod of claim 52 wherein said luminescent compound is a tris-4,7-diphenyl-1, 10-phenanthroline ruthenium (II) salt.
 58. The method ofclaim 57 wherein said luminescent compound is tris-4, 7-diphenyl-1,10-phenanthroline ruthenium (II) chloride.
 59. The method of claim 52wherein said luminescent compound is a tris-2, 2′-bipyridyl ruthenium(II) salt.
 60. The method of claim 59 wherein said luminescent compoundis tris-2, 2′-bipyridyl ruthenium (II) chloride hexahydrate.
 61. Themethod of claim 52 wherein said luminescent compound is 9, 10-diphenylanthracene.
 62. The method of claim 52 wherein, in step (i), thesolution is also contacted with an effective concentration of one ormore biomaterials.
 63. The method of claim 62 wherein said biomaterialis MATRIGEL®.
 64. The method of claim 52 wherein, in step (i), thesolution is also contacted with an effective concentration of one ormore extracellular matrices.
 65. The method of claim 64 wherein saidextracellular matrix protein is collagen.
 66. The method of claim 52wherein, in step (i), the solution is contacted with an effectiveconcentration of one or more additives or coating substances.
 67. Amethod for determining the effects of at least one drug or toxin onrespiring eukaryotic cells comprising: (i) preparing a liquid mediabroth of said eukaryotic cells; (ii) placing said broth in a containerin which said broth is substantially isolated from atmospheric oxygenand placing within said container, but not in direct contact with saidbroth, a sensor composition which comprises a luminescent compound thatexhibits a change in luminescent property, when irradiated with lightcontaining wavelengths which cause said compound to luminesce, uponexposure to oxygen, wherein the presence of the sensor composition isnon-destructive to the eukaryotic cells; (iii) admixing with said liquidmedia broth a quantity of said drug, toxin or chemical; (iv) irradiatingsaid sensor composition with light containing wavelengths which causesaid luminescent compound to luminesce; (v) measuring or visuallyobserving the change in luminescent property from said luminescentcompound while irradiating said sensor compound with said light; and(vi) comparing said measurement to that of a control wherein saidcontrol is selected from the group consisting of: a reagent control notin contact with respiring eukaryotic cells or the drug, toxin orchemical; a reagent control in contact with respiring eukaryotic cellsbut not in contact with the drug, toxin or chemical and a calculatedthreshold, wherein a change in luminescent property relative to thecontrol is indicative of cytotoxicity of the quantity of drug, toxin orchemical to the eukaryotic cells; and (vii) in the event that no suchchange is measured or observed, repeat steps (iv), (v) and (vi), asneeded, to determine the effects of the drug, toxin or chemical on therespiring eukaryotic cells.
 68. The method of claim 67 wherein saidluminescent compound is contained within a matrix which is relativelyimpermeable to water and non-gaseous solutes, but which has a highpermeability to oxygen.
 69. The method of claim 68 wherein said matrixis a rubber or plastic matrix.
 70. The method of claim 68 wherein saidmatrix is a silicone rubber matrix.
 71. The method of claim 68 whereinsaid luminescent compound is adsorbed on solid silica particles.
 72. Themethod of claim 67 wherein said luminescent compound is a tris-4,7-diphenyl-1, 10-phenanthroline ruthenium (II) salt.
 73. The method ofclaim 72 wherein said luminescent compound is tris-4, 7-diphenyl-1,10-phenanthroline ruthenium (II) chloride.
 74. The method of claim 67wherein said luminescent compound is a tris-2, 2′-bipyridyl ruthenium(II) salt.
 75. The method of claim 74 wherein said luminescent compoundis tris-2, 2′-bipyridyl ruthenium (II) chloride hexahydrate.
 76. Themethod of claim 67 wherein said luminescent compound is 9, 10-diphenylanthracene.
 77. The method of claim 67 wherein, in step (ii), aneffective concentration of one or more biomaterials is also placed insaid container.
 78. The method of claim 77 wherein said biomaterial isMATRIGEL®.
 79. The method of claim 67 wherein, in step (ii), aneffective concentration of one or more extracellular matrices is alsoplaced in said container.
 80. The method of claim 79 wherein saidextracellular matrix is collagen.
 81. The method of claim 67 wherein, instep (ii), an effective concentration of one or more additives orcoating substances is also placed in said container.
 82. A method forquantifying respiring eukaryotic cells in a solution comprising: (i)placing said solution in a container in which said fluid issubstantially isolated from atmospheric oxygen and placing within saidcontainer, but not in direct contact with said fluid, a sensorcomposition which comprises a luminescent compound that exhibits achange in luminescent property, when irradiated with light containingwavelengths which cause said compound to luminesce, upon exposure tooxygen, wherein the presence of the sensor composition is nondestructiveto the eukaryotic cells; (ii) irradiating said sensor composition withlight containing wavelengths which cause said luminescent compound toluminesce; (iii) measuring or visually observing the change inluminescent property from said luminescent compound while irradiatingsaid sensor compound with said light; (iv) comparing said measurement tothat of a control not containing respiring eukaryotic cells, whereinsaid control is selected from the group consisting of: a reagent controlnot in contact with respiring eukaryotic cells and a calculatedthreshold, wherein a change in luminescent property relative to theluminescent property of the control is indicative of the presence ofrespiring eukaryotic cells; and (v) in the event that no such increaseis measured or observed, repeat steps (ii), (iii), and (iv) as needed,to quantify respiring eukaryotic cells in said solution.
 83. The methodof claim 82 wherein said luminescent compound is contained within amatrix which is relatively impermeable to water and nongaseous solutes,but which has a high permeability to oxygen.
 84. The method of claim 83wherein said matrix is a rubber or plastic matrix.
 85. The method ofclaim 83 wherein said matrix is a silicone rubber matrix.
 86. The methodof claim 83 wherein said luminescent compound is adsorbed on solidsilica particles.
 87. The method of claim 82 wherein said luminescentcompound is a tris-4, 7-diphenyl-1, 10-phenanthroline ruthenium (II)salt.
 88. The method of claim 87 wherein said luminescent compound istris-4, 7-diphenyl-1, 10-phenanthroline ruthenium (II) chloride.
 89. Themethod of claim 82 wherein said luminescent compound is a tris-2,2′-bipyridyl ruthenium (II) salt.
 90. The method of claim 89 whereinsaid luminescent compound is tris-2, 2′-bipyridyl ruthenium (II)chloride hexahydrate.
 91. The method of claim 82 wherein saidluminescent compound is 9, 10-diphenyl anthracene.
 92. The method ofclaim 82 wherein, in step (i), the solution is also contacted with aneffective concentration of one or more biomaterials.
 93. The method ofclaim 92 wherein said biomaterial is MATRIGEL®.
 94. The method of claim82 wherein, in step (i), the solution is also contacted with aneffective concentration of one or more extracellular matrices.
 95. Themethod of claim 94 wherein said extracellular matrix is collagen. 96.The method of claim 82 wherein, in step (i), the solution is contactedwith an effective concentration of one or more additives or coatingsubstances.